Ischemic cardiac disease and peripheral vascular disease are major health problems affecting hundreds of millions of people worldwide. Ischemia results when there is a lack of oxygen supply. It is estimated that about half of the deaths that occur in the United States each year alone are caused by ischemic heart disease. This invention relates, in part, to methods for the treatment of such diseases and pharmaceutical compositions in the treatment thereof.
Oxygen is essential for an organism's survival, given its role in essential metabolic processes including oxidative phosphorylation in which O2 serves as electron acceptor during ATP formation. Tissue damage can result from hypoxia, that is, when oxygen supply in tissue is insufficient to meet metabolic demands. Hypoxia can be caused by various medical conditions, including atherosclerosis, chronic illness, trauma, and surgical procedures. Accordingly, hypoxia plays an important role in the pathogenesis of major causes of mortality, including cancer, cerebral and myocardial ischemia, and chronic heart and lung diseases.
Organisms can sense O2 concentration and adaptively respond to hypoxia. These adaptive responses either increase O2 delivery or activate alternative metabolic pathways that do not require O2. There are a number of hypoxia-inducible gene products that participate in these responses. Included, are genes that code for erythropoietin (hereinafter “EPO”), vascular endothelia growth factor (hereinafter “VEGF”), tyrosine hydroxylase, and glycolytic enzymes. See Bunn, H. F & Poyton, R. O., “Oxygen sensing and molecular adaptation to hypoxia”, Physiol. Rev., Vol. 76, pp. 839-885 (1996); Semenza, G. L., “Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1”, Annu. Rev. Cell. Dev. Biol., Vol. 15, pp. 551-578 (1999); Shweiki, D., et al., “Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis”, Proc. Natl. Acad. Sci. U.S.A.., Vol. 92, pp. 768-772 (1995). The transcriptional regulator hypoxia-inducible factor 1 (hereinafter “HIF-1”) is an essential mediator of O2 homeostasis and regulates the transcription rate of many genes including the aforementioned genes. See Wang, G. L., et al., Biochem. Biophys. Res. Commun., Vol. 86, pp. 15-22 (1995). The number of target genes activated by HIF-1 includes genes whose protein products are involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability, vascular remodeling, and vasomotor responses. Semenza, G. L., “HIF-1: mediator of physiological and pathophysiological responses to hypoxia,” J. Appl. Physiol. Vol. 88, pp. 1474-1480 (2000); Semenza, G. L., “Hypoxia-inducible factor 1: master regulator of O2 homeostasis,” Genetics & Development, Vol. 8, pp. 588-594 (1998).
Structurally, HIF-1 is a heterodimer of two subunits, HIF-1α and HIF-1β. The biological activity of HIF-1 is determined by the expression and activity of the HIF-1α subunit. See Jiang, B-H, et al., “Transactivation and inhibitory domains of hypoxia-inducible factor 1α: modulation of transcriptional activity by oxygen tension”, J. Biol. Chem., Vol. 272, pp. 19253-60 (1997). The in vivo regulation of HIF-1α biological activity occurs at multiple levels, including mRNA expression, protein expression, nuclear localization, and transactivation. Semenza, J. Appl. Physiol., Vol. 88, page 1476 (2000). Hypoxia, in turn, is known to have at least two independent effects on HIF-1α activity: (1) hypoxia increases the steady-state levels of HIF-1α protein by stabilizing it (i.e., decreasing its degradation); and (2) hypoxia increases the specific transcriptional activity of the protein (i.e., independent of the protein concentration). Jiang, B. H., et al., “Dimerization, DNA binding, and transactivation properties of hypoxia-inducible factor 1,” J. Biol. Chem., Vol. 271, pp. 17771-78 (1996). Given HIF-1's role in hypoxia, treatments utilizing HIF-1 in the treatment of hypoxia-related disorders, have been described. For example, U.S. Pat Nos. 5,882,914; 6,020,462; 6,124,131 and international publication number WO 00/10578.
Although the known number of target genes activated by HIF-1 continues to increase, the role of HIF-1 in the activation of VEGF gene transcription in hypoxic cells is well established. Semenza, J. Appl. Physiol. Vol. 88, page 1477 (2000). VEGF itself mediates a number of responses including vasodilation, vascular permeability, and endothelial cell migration and proliferation through receptors that are restricted to vascular endothelium and certain hematopoietic cells. The combined effects of VEGF are important to the promotion of an angiogenic response. The restricted localization of VEGF receptors provides a level of specificity that makes VEGF an important target for angiogenic therapy. For example, the promotion of blood vessel growth has been demonstrated in animal models of coronary and limb ischemia. See Pearlman, J. D., et al., “Magnetic resonance mapping demonstrates benefits of VEGF-induced myocardial angiogenesis”, Nat. Med. Vol. 1, pp. 1085-1089 (1995); Takeshita, S., et al., “Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model”, J. Clin. Invest., Vol. 93, pp. 662-670 (1994). There are several clinical trials in progress to assess the efficacy of both exogenously administered VEGF protein as well as expression vectors for the VEGF gene. See Hendel, R. C., et al., “Effect of intracoronary recombinant human vascular endothelial growth factor on myocardial perfusion—Evidence for a dose-dependent effect”, Circulation, Vol. 101(2), pp. 118-121 (2000); Schwarz, et al., “Evaluation of the effects of intramyocardial injection of DNA expressing vascular endothelial growth factor in a myocardial infarction model in the rat—Angiogenesis and angioma formation”, J. Amer. Coll. Cardiol., Vol. 35(5), pp. 1323-1330 (2000).
Another approach to utilizing the effects of VEGF in proangiogenic therapy is to stimulate its production from the tissues needing new vessels. Secretion of VEGF appears to be dependent on its rate of biosynthesis since the intracellular storage of VEGF protein has not been demonstrated. The biosynthesis of VEGF is primarily controlled by regulating the amount of VEGF mRNA. See Shweiki, D., et al, “Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis”, Nature, Vol. 359, pp. 843-845 (1992). In turn, the amount of mRNA is controlled by activation of transcription through regulatory elements located in the 5′ promoter sequence of the VEGF gene as well as by less characterized mechanisms that stabilize VEGF mRNA. See Levy, A. P., et al., “Transcriptional regulation of the rat vascular endothelial growth factor gene by hypoxia”, Drug Discov. Today, Vol. 270, pp. 13333-13340 (1995); Ikeda, E., et al., “Hypoxia-induced transcriptional activation and increased mRNA stability of vascular endothelial growth factor in C6 glioma cells”, J. Biol. Chem., Vol. 270, pp. 19761-19766 (1995); Levy, A. P., et al., “Post-transcriptional regulation of vascular endothelial growth factor by hypoxia”, Drug Discov. Today, Vol. 271, pp. 2746-2753 (1996).
Various treatments using VEGF have been suggested (e.g., U.S. Pat. No. 5,073,492 issued Dec. 17, 1991; U.S. Pat. No. 5,194,596 issued Mar. 16, 1993; and U.S. Pat. No. 5,219,739 issued Jun. 15, 1993) for ameliorating conditions such as myocardial infarction, diabetic ulcers, and surgical wounds. In particular, several small molecules have been described which mimic the hypoxic induction of VEGF by activating HIF-1α. However, many of these molecules, such as cobaltous chloride or deferoxamine cannot be considered candidate drug-like molecules because of unfavorable pharmacokinetic characteristics. Still other molecules, such as mersalyl, cannot be considered because of their reactivity. See Agani, F. & Semenza, G. L., “Mersalyl is a novel inducer of vascular endothelial growth factor gene expression and hypoxia-inducible factor 1 activity”, Mol. Pharmacol., Vol. 54, pp. 749-754 (1998). Although several growth factors, such as platelet derived growth factor-BB, transforming growth factor β1, and hepatocyte growth factor, have also been shown to induce VEGF, their effects may be limited to certain tissue types and transformed cell lines and therefore are probably not mediated through HIF-1α. See Brogi, E., et al., “Indirect angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor; the case for paracrine amplification of angiogenesis”, Circulation, Vol. 90, pp. 649-652 (1994); Van, B. E., et al., “Potentiated angiogenic effect of scatter factor/hepatocyte growth factor via induction of vascular endothelial growth factor: the case for paracrine amplification of angiogenesis”, Circulation, Vol. 90, pp. 381-390 (1998). Therefore, there exists a continuing need to identify classes of compounds that induce VEGF at the transcriptional level to increase vascularization of afflicted tissue for the treatment of the aforementioned disorders.
HIF-1 is a transcription factor that also regulates the hypoxia-inducible EPO gene. HIF-1 binding is required for EPO transcriptional activation in response to hypoxia. Semenza, G. L.,“Regulation of erythropoietin production: New insights into molecular mechanisms of oxygen homeostasis”, Hematol. Oncol. Clin. North Am., Vol. 8, pp. 863-884 (1994). In particular, HIF-1α binds to the 3′ hypoxia-response element of the EPO gene which results in the marked enhancement of EPO transcription. Semenza, G. L., et al. “Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1”, J. Biol. Chem., Vol. 269, pp. 23757-63 (1994). EPO, in turn, is essential for maintenance of red blood cells by controlling the proliferation and differentiation of erythroid progenitor cells into red blood cells. Krantz, S. B., “Erythropoietin,” Blood, Vol. 77, pp 419-434 (1991). During fetal development, the liver serves as the primary source of EPO. Shortly before birth, production of EPO in the liver decreases and the kidney becomes the primary source of EPO. However, in adults other organs such as the liver and brain produce small but significant amounts of EPO. A erythropoietin deficiency is associated with anemia. In humans, the most prevalent form of anemia is associated with kidney failure.
Compounds have been described that enhance the biosynthesis of EPO such as those described in U.S. Pat. No. 5,985,913 issued Nov. 16, 1999. Another approach is using injectable recombinant EPO, which is currently the therapy of choice for the treatment of anemia due to chronic renal failure. EPO has been described in the treatment of anemia: associated with chemotherapy; that occurs as a consequence of AIDS; and due to prematurity and autologous blood donation. EPO has even been suggested as a general use agent in pre-operative elective surgery. However, its extensive use could be limited by high production costs and lack of oral bioavailability. See Qureshi, S. A., et al., “Mimicry of erythropoietin by a nonpeptide molecule”, PNAS, Vol. 96(21) pp. 12156-61 (1999). Therefore, there exists a continuing need for the development of classes of molecules that increase endogenous EPO at the transcriptional level for the treatment of the aforementioned disorders.
Thus, it would be advantageous to identify a class of compounds that are effective in treating hypoxia related disorders.